Tubular prostheses

ABSTRACT

Tubular prostheses are provided for use in airways, upper digestive, and urinary tracts. Each of these uses has its own specific sets of biological specifications, based on what it must contain and exclude and the physical and chemical pressures and stresses to which it is subjected. The prostheses may be made from allogeneic cells. Thus they can be manufactured and stored prior to an individual&#39;s personal need arising.

TECHNICAL FIELD OF THE INVENTION

This invention is related to the area of artificial replacements fordiseased or damaged anatomical conduits. In particular, it relates toconduits of fluids, solids, and gasses.

BACKGROUND OF THE INVENTION

Currently, there are about 2,000 patients per year in the U.S. who needreplacement tracheal tissue. Causes for this include tracheal cancer,invasive infections of the trachea or bronchi, and trauma. There are noreplacements currently available fir trachea in humans. At best, when asegment of trachea is resected, the only surgical option is to “pulltogether” the two ends of the trachea and sew them together, hoping thatthe anastomosis does not “pull apart” thereafter.

Currently in the U.S., approximately 4,000 patients per year need anesophageal replacement. This is due primarily to esophageal cancer,though trauma and infection are causes a small number of cases ofesophageal replacement. Currently, there is no available replacement foresophageal tissue. What is done currently to replace esophagus is one oftwo procedures. Either a segment of the stomach is loosened from itsconnections in the abdomen and brought up into the chest, to anastomoseto the remnant esophagus; or, a segment of large bowel (i.e., colon) isresected from the patient and sewn in to replace the resected esophagealtissue. Both of these procedures have many complications and a viableesophageal replacement is certainly medically needed.

Every year in the U.S., approximately 10,000 patients undergocystectomy, and require a urinary conduit to drain urine outside thebody [Healthcare Cost and Utilization Project, N.I.S., 2007.]. In almostall cases, bowel is harvested from the patient to form either anoncontinent urinary diversion, or a continent urinary diversion that iscatheterized intermittently to drain urine through a continent stoma.[Konety, B. R., Joyce, G. E., Wise, M., Bladder and upper tracturothelial cancer. Journal of Urology, 2007. 177: p. 1636-1645.]. Due tosurgical simplicity and lower complication rates, creation of anoncontinent urinary conduit is the most common approach for drainingurine following cystectomy. Most typically, a 15-25 cm length of ileumis harvested from the patient for use as the urinary conduit, and theremaining bowel is reanastomosed [Gudjonsson, S., Davidsson, T.,Mansson, W., Incontinent urinary diversion. BTU International, 2008.102: p. 1320-1325.]. One end of the harvested ileal segment isanastomosed to the patient's ureters, and the other end is then broughtout to the skin to form a stoma through which urine can drain.

Though widely used, ileal conduits pose many problems that can lead toshort-term and long-term complications [Konety, Allareddy, V., Influenceof post-cystectomy complications on cost and subsequent outcome. Journalof Urology, 2007. 177: 280-287.]. In the short term, patients may sufferfrom complications at the bowel harvest site, including anastomoticleaks and peritonitis. In addition, ileal urinary conduits may sufferfrom ischemia and necrosis, which can lead to perforation, anastomoticbreakdown, and leakage of urine from the conduit. In the long term, manypatients suffer from chronic hyperchloremic metabolic acidosis, due toresorption of urine electrolytes through the conduit wall. Since ilealconduits harbor bacteria, patients also commonly suffer from recurrenturinary tract infections and pyelonephritis, as bacteria from theconduit infect the more proximal urinary system. Hence, there is asignificant medical need for an improved method for urinary diversion,that avoids many of the complications associated with the use of Healconduits [Dahl, D. M., McDougall, W. S., Campbell-Walsh Urology, 9thEdition: Use of intestinal segments and urinary diversion, ed. A. J.Wein, Kavoussi, Novick, A. C. 2009].

There is a continuing need in the art tier replacements for theseimportant conduits, as well as other tubular tissues in the body, suchas ureters, urethras, intestine, etc.

SUMMARY OF THE INVENTION

According to one aspect of the invention an artificial airway isprovided for replacement of damaged or diseased tissue by implantationinto a respiratory tract of a recipient. The artificial airway comprisesa tubular stent and substantially acellular, non-layered, contiguous,extracellular matrix surrounding the stent on its inner and outersurfaces.

According to another aspect of the invention a method is provided ofmaking an artificial airway. A tubular stent having an inner and outersurface is encased in at least two layers of a mesh scaffold. A first ofthe two layers is on the interior surface, and a second of the twolayers is on the exterior surface. The mesh scaffold is seeded withvascular smooth muscle cells, which are then cultured on the meshscaffold in a bioreactor for 6-10 weeks. The smooth muscle cellsproliferate and secrete extracellular matrix on the mesh scaffold. Thetubular stent is decellularized to form an acellular tubular airwaystent encased in extracellular matrix on the inner and outer surfaces.

Another aspect of the invention is an artificial esophagus forreplacement of damaged tissue by implantation. The esophagus comprisessubstantially acellular extracellular matrix formed as a tube of greaterthan 10 mm diameter. The artificial esophagus has a suture retention ofgreater than 150 grams.

Yet another aspect of the invention is an artificial urinary conduit forimplantation in a patient in need of urinary diversion and drainage. Theconduit comprises a tubular, substantially acellular, extracellularmatrix formed as a tube of greater than 10 mm diameter. Theextracellular matrix is produced and secreted by non-autologous smoothmuscle cells. The artificial urinary conduit has a rupture strength ofgreater than 1000 mm Hg.

Yet another aspect of the invention is an artificial urinary conduit forimplantation in a patient in need of urinary diversion and drainage. Theconduit comprises a tubular, substantially acellular, extracellularmatrix formed as a tube, as well as a tubular stent. The extracellularmatrix is produced and secreted by non-autologous smooth muscle cells.The artificial urinary conduit has a rupture strength of greater than1000 mm Hg.

These and other embodiments which will be apparent to those of skill inthe art upon reading the specification provide the art with new surgicaltools tier repairing damaged anatomical conduits.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: to Schematic of placement of stent with surrounding PGA scaffoldinside of bioreactor, in preparation for culture.

FIG. 2: Seeding of smooth muscle cells (SMCs) onto PGA mesh encasingmetal stent in the bioreactor.

FIG. 3A-3B: Gross photos of engineered trachea. Smooth muscle cellscultured on PGA mesh surrounding a metal stent. After 8 weeks ofculture, cells are removed by decellularization, leaving behind theengineered extracellular matrix encasing the metal stent. Thisengineered trachea is 1.6 cm in diameter and approximately 8 cm inlength. FIG. 3A: photograph of outside of engineered trachea, showingsmooth tissue covering entire external surface. FIG. 3B: photograph ofinside of engineered trachea, showing smooth tissue covering entireinner surface.

FIG. 4: Gross photo of engineered trachea, showing water-tight tissue.Engineered trachea is 1.6 cm in diameter and 8 cm in length. Theengineered trachea, consisting of decellularized tissue encasing a metalstent, is filled with colored liquid and held aloft. The acellulartissue, which envelopes the walls and end of the stent, is sufficientlyrobust to hold liquid, as the visible level of red liquid inside thetrachea, demonstrates (arrow).

FIG. 5: Collagen content of engineered trachea tissues: Biochemicalassay for hydroxyproline shows collagen content as a fraction of drytissue weight, before and after decellularization. Increase in collagenas a fraction of dry weight after decellularization indicates thatcellular material is removed, while collagenous exxtracellular matrixremains.

FIG. 6: Schematic of culture of engineered esophagus in bioreactor.

FIG. 7: Engineered acellular esophagus held with forceps. Segment ofengineered esophagus is 1.5 cm in diameter by 5 cm in length. It isstrong enough to hold retraction by two sets of forceps, shown.Esophagus is shown by arrows on either side.

FIG. 8A-8C: Histologies of engineered esophagus. FIG. 8A: H&E stain ofengineered esophagus shows cellular nuclei (purple) and extracellularmatrix (pink). Scale bar is 50 microns. FIG. 8B: Masson's trichromestain shows blue collagen in engineered esophagus, nuclei of cellsappear red. Scale bar is 50 microns. FIG. 8C: Masson's trichrome stainof decellularized engineered esophagus, red nuclei are absent indicatingloss of cells. Scale bar=50 microns.

FIG. 9: Suture retention strength of engineered esophagus. Sutureretention in grams is shown before decellularization, and afterdecellularization regimens lasting either 0.45 or 80 minutes. After 0.45minutes of decellularization, suture retention strength is greater than100 grams. This is indicative of an implantable engineered tissue (Dahl,et al, Science Translational Medicine 3:68pc2, 2011).

FIG. 10: Collagen content of engineered esophagus. Hydroxyprolinecontent of engineered esophagus before decellularization, and after 45and 80 minutes of decellularization. Progressive decellularizationremoves more cellular material, thereby increasing the percentage ofcollagen remaining in the tissue as a fraction of dry weight.

FIG. 11: Anatomy of ileal conduit implantation. Ureters are sewn to asegment of ileum, which is brought to the skin as a stoma.

FIG. 12A-12C: Acellular engineered conduits. FIG. 12A shows a grossphoto of a 6-mm conduit after harvest from bioreactor anddecellularization. FIG. 12B shows scanning electron microscopy image ofan acellular conduit, showing smooth luminal surface and pores withinthe wall. FIG. 12C shows conduit pressurized to 100 mm Hg, which showsno leaks to liquid and an excellent kink radius of no greater than 1.5cm.

FIG. 13A-13C: FIG. 13A: gross photograph of aorto-caval graft in ababoon; FIG. 13B: immunostaining for alpha-actin, a smooth musclemarker, shows smooth muscle cells infiltrating into the graft (g); FIG.13C: T-cell proliferation in response to graft conduit is less than tocontrol, teflon.

FIG. 14A-14G: Schematic drawings showing the growth and development ofcells and matrix about a stent FIG. 14A shows a bioresorbable meshscaffold surrounding a stent on its external surface. FIG. 14B shows abioresorbable mesh scaffold on the interior surface of a stent. FIG. 14Cshows a bioresorbable mesh scaffold placed on both the interior and theexterior surfaces of the stent, FIG. 141) shows the two layers describedin FIG. 14C which have been stitched together to unify the two layers.FIG. 14E shows cells (white) which have been seeded on the mesh. FIG.14F shows matrix (grey) which has been synthesized and secreted by theseeded cells. FIG. 14G shows an acellular matrix enveloping the stentafter the cells have been removed by decellularization process.

FIG. 15: Neck of rat open, with native trachea cut and two cartilagenousrings removed. Engineered trachea is anastomosed to proximal trachealtissue, with distal end of engineered trachea extending upward.

FIG. 16: Engineered human, decellularized trachea implanted into ratrecipient. Native and engineered tracheal tissues are indicated infigure.

FIG. 17: H&E stain of tissue that has grown from host airway inside ofthe engineered trachea after 2 weeks of implantation. White arrowheadspoint out red blood cells in capillaries in the tissue ingrowth,indicating extensive microvascularization of ingrown tissue. Scale baris 50 microns.

FIG. 18: Immunostain of tissue ingrown into lumen of engineered tracheaafter 2 weeks of implantation. Blue is DAPI nuclear stain, while red isimmunostain for cytokeratn-14, a marker of tracheal epithelium. Thisimage shows that many of the cells in the lumen of the engineeredtrachea were ingrown epithelium from the recipient, Scale bar 50microns.

FIG. 19: Low-power H&E image of engineered, human decellularized tracheathat was implanted into a nude rat for 6 weeks and then explanted.Surrounding native fibrous tissue is indicated. Locations of struts ofnitinol stent, visible as square holes in the tissue, are indicated withasterisks (*). The implanted engineered matrix has some evidence ofcellular infiltration after 6 weeks of implantation (nuclei visible inbetween struts, and near bottom of image). Neo-tissue in-growth in thelumen of the engineered trachea is visible. Scale bar 20 microns.

FIGS. 20A-20B: A graft was sewn into the esophagus of a pig to replacean excised segment of esophagus (FIGS. 20A-20B), This shows thepotential for full-circumference replacement of esophagus.

FIG. 21: H&E stain of engineered trachea that was implanted and thenexplanted after 6 weeks. Holes in tissue section that contained stentstruts are indicated by black arrows. Clearly, struts of the stent aredensely embedded into the extracellular matrix tissue (pink and orangein the image). An inner tissue layer of epithelium (purple nuclei) hasalso formed in the engineered trachea. Scale bar=1 mm.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have developed new surgical tools for repairing damagedanatomical conduits, including airways, upper digestive tract, andurinary conduits. Because the conduits are formed by seeding cells on atubular substrate, rather than as sheets which are subsequently wound toform a tube, or tubes that are layered, they are not subject to therisks of slippage or leakage can arise with a layered approach.Furthermore, no slippage or leakage occurs between the tissue and thetubular substrate (e.g., stent) because the tubular substrate or stentis integrated within the non-layered tissue. Moreover, there is no riskof unwinding of tissue layers. The conduits form a composite artificialtissue in which, in some embodiments, a stent is totally enveloped andencased within extracellular matrix that has been grown and secreted insitu. The extracellular matrix is a naturally occurring matrix that isproduced by cells. The matrix is, preferably, not a denatured orchemically processes material, such as gelatin (which is denaturedcollagen) or proteins that are cross-linked by artificial processes suchas freezing, or drying, or gluteraldehyde or other chemical fixation.The naturally occurring extracellular matrix that is produced by livingcells is preferable for the practice of the current invention because itis more readily remodeled by host cells after implantation, and becauseit is less likely to induce adverse host responses such as inflammationor calcification as compared to denatured, processed, or artificiallycross-linked extracellular matrices. Because of its mode of manufacture,no gaps exist or form during and after implantation. The conduits are ineffect composite tissues. The tissues may be a composite of cells andmatrix (decellularized), polymer fragments, and an optional stentingmaterial. The extracellular matrix may, for example, bridge stent strutsand completely incorporate the stent material.

The tissues so formed have matrix (ECM) that fits “snugly” around thestent struts. The tissues adjoin, are connected with, abut, are next to,have a common boundary touch, are contiguous with, share a common borderwith the stent. They form a unitary composite tissue that is not subjectto separation and deconvolution into constituent parts.

Cells used in the conduits can be allogeneic, autologous, syngeneic, orxenogeneic. Typically cells used in making the conduits are killedand/or removed prior to use. The killing and/or removal of cellsdiminishes the potential for adverse immune reactions. Killing and/orremoval of cells leaves less than 50%, less than 75%, less than 80%,less than 85%, less than 90%, or less than 95% of the cells viable, asassessed by trypan blue staining, nucleotide incorporation, or proteinsynthesis. Remaining extracellular matrix is highly conserved amongindividuals, and among species, rendering it less likely to provoke anadverse immune reaction than live cells. Vascular smooth muscle cellsare one type of cell that can be used to make the extracellular matrix.These can be isolated from any vasculature of a human or other mammal,including from the aorta. Much of the secreted extracellular matrixcomprises collagen. Collagen may comprises at least 5%, at least 10%, atleast 15%, at least 20%, at least 25%, at least 30% of the extracellularmatrix. Typically the extracellular matrix is grown until it achieves athickness of at least 50 microns, at least 100 microns, at least 150microns, at least 200 microns, at least 250 microns, at least 300microns, at least 400 microns, or at least 500 microns. Diameter of theconduits may be controlled during manufacturing. Typically these mayhave an internal diameter of at least 10, at least 15, at least 20, atleast 25, at least 30, at least 35, 40, at least 45, at least 50 mm.

Under some circumstances, it may be desirable to have live cells on orwithin the conduit. Such cells may be seeded upon the conduit and eithergrown in culture or grown in situ. The cells may be seeded in situ aswell, by endogenous cells of the recipient which migrate and establishthemselves on the artificial prosthesis. The cells may be derived fromthe patient or from another source. The cells may be useful formimicking and recreating natural conditions in the host. Alternativelythe cells may be used as in situ factories to produce a product that isdesirable, such as a growth hormone, chemokine, blood factor, and thelike. Suitable cells for seeding on an airway conduit include withoutlimitation tracheal cells such as epithelial cells, cartilage cells,endothelial cells, smooth muscle cells, and fibroblasts.

A stent for use in an airway prosthesis may be made of metal, a polymer,or other natural or artificial biocompatible substance. The stent may benon-degradable, or may be degradable. These stents typically have aperforated structure, which permits attachment of inner and outer layersof substrate, for example, mesh or fabric. In some embodiments, thestent is provided with mesh only on the outer surface of the stent,thereby allowing cells to grow and extracellular matrix on the outsideand then subsequently completely envelope the struts of the stent. Inother embodiments, the mesh is applied only to the inside surface ofstent, which after cell seeding also allows the cells to envelope boththe mesh and the struts of the stent with both cells and extracellularmatrix. One suitable mesh which can be used as a substrate for cellgrowth is made of polyglycolic acid. Typically, during culturingpolyglycolic acid mesh degrades spontaneously, and fragments of it arewashed away in the culture medium or are phagocytosed by the culturedcells and degraded. Some fragments may remain. Other biodegradable onnon-degradable substrates can be used as are known in the art, such aspolylactic acid, polycaprolactone, polyanhydrides, polyethylene glycol,as well as other biocompatible polymeric substances. Other biocompatiblesubstrates include collagen, gelatin, elastin, cellulose, alginate, andother substances which support the growth of cells in culture.Substrates may be in a mesh format, or may take the form of a gel or asponge.

While the conduits are described as tubular, they may also contain oneor more branches, so that the conduit is in the shape of for example, aY, X, T, or F. Conduits with such branches are considered tubular, aswell. The conduits described may be implanted to replace, line,reinforce, or by-pass an existing physiological or implanted conduit.

Conduits which are made by growing cells on a tubular stent may have anadditional advantage over conduits formed using rolled sheets inside andoutside of a stent. The conduits made by growing cells may have theextracellular matrix rotationally fixed with respect to the stent. Theinner and outer surfaces of extracellular matrix may additionally berotationally fixed with respect to one another. Being so fixed, slippageand leakage is minimized. The two surfaces may be so fixed by, forexample, interlacing the substrates upon which the cells secreting theextracellular matrix are grown. The cells and extracellular matrix may,for example, envelope or bridge the stent struts, and may completelyincorporate the stent material.

Conduits that are grown by culturing cells on a substrate that encases atubular stent, either on the inside or the outside or on both sides,have the advantage of being comprised of a single tissue that envelopesand encases the stent material. The resulting material is a truetissue-stent composite material. This configuration has many functionaladvantages over earlier systems that involve rolling a sheet of tissuearound the inside or outside of a stent. For example, in situationswhere tissue is rolled around the stent, it can occur that the sheets oftissue do not fuse with each other, or do not fuse with the stentmaterial. This lack of fusion of tissue sheets results in a construct inwhich pieces of tissue and stent material can slip relative to oneanother, resulting in a conduit which is structurally unstable Iiicontrast, by culturing cells on a scaffold which fully encases the stentmaterial, the resulting conduit is comprised of a single piece oftissue-stent composite material, and contains no sheets of tissue whichmay move or slip relative to one another. Such conduits are then morehighly suited to various applications wherein the conduit must beliquid-tight or air-tight. In addition, such conduits are more highlysuited to serving as replacements for native tubular tissues, such astrachea, bronchus, intestine, esophagus, ureter, urinary conduit, orother tubular tissues which must function to contain liquid or air orboth. In contrast, stents that are wrapped with sheets of exogenoustissue may be poorly suited for these applications, since the slippageof tissues and stent material can cause leakage of air or fluid. Inaddition, a single tissue-stent composite material displays superiorhandling properties for surgical implantation, in contrast to wrappedtissue sheets which can slide and become detached from the stentmaterial. In addition, a single tissue-stent composite material willwithstand physiological stresses following implantation, such aspressurization, shear forces, fluid flow, and the like, while a stentencased in tissue sheets may delaminate and lose structural integrityupon exposure to physiological forces in the body.

Decellularization of the conduit may involve the killing and/or removalof cells from a scaffold or substrate. Any means known in the art may beused, including but not limited to the use of agitation and the use ofdetergents. The decellularization process must be balanced between thelimits of being sufficiently harsh to kill or dislodge the cells andsufficiently gentle to maintain the extracellular matrix structureintact. Substantially acellular extracellular matrix remains after thedecellularization process. The prosthesis contains less than 50%, lessthan 75%, less than 80%, less than 85%, less than 90%, or less than 95%of the cells viable, as assessed by trypan blue staining, BrdUnucleotide incorporation, TUNEL staining, or protein synthesis.

Prostheses or conduits may be stored prior to implantation in arecipient mammal. The storage may occur before or afterdecellularization has occurred. Storage may be at various temperatures,but typically will be at or below 4 deg C., 0 deg C., −20 deg C., −40deg C., or −60 deg C. Storage may be for at least hours, at least days,at least weeks, at least months or at least years. In addition, conduitsmay be stored at room temperature, at or below 20 deg C., 25 deg C., 30deg C., 35 deg C., or 40 deg C. In general, it is not desirable to storethe conduits at temperatures above 40 deg C.

Trachea serves the function of conducting humidified air from theoutside into the lungs. To serve this purpose, a trachea replacementtissue must be able to withstand compressive pressures and negativeintra-luminal pressure. This is because, every time we take an inhaledbreath, we exert a negative pressure on the tracheal lumen. Thus, toremain patent during inspiration, it is necessary for a trachea to beinvested with some sort of “stenting” function that prevents collapse.In the native trachea, this function is sub-served by rings of cartilagetissue that are embedded in the wall of the trachea. In the engineeredtrachea (conduit), this stenting function is served by a metal (or othermaterial) stent that is physically embedded inside an engineered tissue.

Another key aspect of functional tracheal tissue is the ability toremain air- and liquid-tight. This is because air that we inhale isfilled with particles and micro-organisms. If micro-organism-containingair were allowed to penetrate into the mediastinum of the patient, thenthis would result in a mediastinal infection that would cause excessivemorbidity/mortality. Hence, any replacement trachea should be “tight” toliquid and air in order to be function, From the data shown below, it isapparent that the engineered trachea is water-tight. In addition, animalimplantation studies of engineered trachea show that the tracheamaintains patency during respiration, and does not leak either liquid orair, does not develop infection or perforation, and adequately serves asa conduit to conduct air to the lungs of the animal.

Another function of trachea is to produce mucous which protects the wallof the trachea from invading organisms and excessive dehydration. Thismucous is generally produced by mucous-producing cells on the lumen ofthe trachea. Our current engineered trachea consists solely ofdecellularized matrix that envelopes a metal stent. After implantation,the engineered trachea re-populates with native tracheal epithelialcells from the recipient, as shown in the drawings. By becoming denselyinvested with native cells of the trachea, the implanted and initiallyacellular trachea becomes more physiologically functional.Alternatively, it may be possible to “pre-coat” the engineered tracheawith autologous epithelial cells prior to implantation.

One advantage of the tissue-stent composite trachea is that the stentmaterial is encased in tissue and is therefore shielded from particlesand micro-organisms that are inhaled during respiration. This, in turn,results in resistance to infection of the stent material, orcolonization with bacteria, fungi, or other inhaled micro-organisms. Bycompletely encasing the stent material, predisposition to stentinfection is minimized and hence function is enhanced.

The engineered trachea may be comprised of a stent (which can be made ofmetal, although it could be made of any biocompatible stenting material,such as a degradable or non-degradable polymer), around which iscultured a layer of vascular smooth muscle cells. To culture the cellson the stent, one or two layers of degradable polymer scaffold made of,for example, polyglycolic acid (PGA) mesh can be wrapped around thestent—one on the outside of the stent, one on the inside, or just onelayer on one side of the stent, depending upon the usage andapplication. The layers of PGA mesh scaffold can then be interwoven toproduce an encasement of the metal stent within two layers of PGA mesh.Alternatively, the stent can be manufactured so as to contain the meshscaffold and the stent-scaffold material is produced as a single,composite piece. The sterilized stent-mesh composite can then be seededwith vascular smooth muscle cells. Airway smooth muscle is an importantcomponent of all large airways, including human trachea. The cells maybe seeded onto the PGA mesh scaffold and cultured for 6-10 weeks inspecialized growth media within a bioreactor. During this time, thesmooth muscle cells can proliferate on the PGA mesh and secreteextracellular matrix, composed mainly of collagen but also containingother substances such as glycosaminoglycans, fibronectin, vitronectin,elastin, and other extracellular matrix molecules. At the conclusion ofculture, the metal stent is then encased in engineered tissue comprisedof smooth muscle cells, extracellular matrix, and PGA polymer fragments.The tissue can then be decellularized, to produce a substantiallyacellular, engineered trachea that is non-living and can be stored forseveral months in buffer solution. This structure should benon-immunogenic when implanted into any allogeneic (i.e., same species)recipient as the source of the smooth muscle cells.

Esophagus has several key functions in the body. First and mostimportantly, it must provide an air-tight and water-tight conduit thatprevents the leakage of food into the surrounding mediastinum. Since allof the food and drink that we consume is contaminated with bacteria, itis essential that the esophagus retain all food material and prevent itfrom entering the chest/mediastinum, where it would cause infection withsignificant attendant morbidity/mortality. Additionally, it mayimpermeable to gas.

A second key function of the esophagus is to provide peristalsis, orrhythmic contractility, to force food from the upper esophagus into thestomach. To perform this function, the esophagus may be comprised mainlyof intestinal smooth muscle that has rhythmic contractile capability.Other cells which may populate the artificial esophagus include suchesophageal cells as epithelial cells, endothelial cells, smooth musclecells, and fibroblasts. To maintain adequate tensile strength andprevent tearing, the esophagus also may have significant collagenousextracellular matrix. An esophagus will typically have a sutureretention of greater than 100 grains, greater than 125 grams, greaterthan 150 grams, greater than 175 grams, greater than 200 grams, orgreater than 225 grams. Its rupture strength may be greater than 500 mmHg, greater than 750 mm Hg, greater than 1000 mm Hg, greater than 1250mm Hg, greater than 1500 mm Hg, or greater than 2000 mm Hg. Unlike thetracheal prosthesis, the esophagus may not require any “stentingfunction” to maintain patency.

Our tissue engineered esophagus consists of an engineered tissue that ismade from vascular smooth muscle cells that are cultured on a degradablepolymer scaffold made of PGA. After 6-10 weeks of culture, theengineered tissue is decellularized, to produce a substantiallyacellular engineered esophagus that can be stored on the shelf formonths at a time.

Urinary conduits may be transplanted into a recipient, such as a humanpatient, connecting one or both ureters of the patient and drainingthrough a stoma in the skin, or replacing a segment of ureter orurethra. A urinary conduit will typically have a suture retention ofgreater than 100 grams, greater than 125 grams, greater than 150 grams,greater than 175 grams, greater than 200 grams, or greater than 22.5grams. Its rupture strength may be greater than 500 mm Hg, greater than750 mm Hg, greater than 1000 mm Hg, greater than 1250 mm Hg, greaterthan 1500 mm Hg, or greater than 2000 mm Hg. After implanting, a urinaryconduit may be populated with endogenous cells such as urinaryepithelial cells, endothelial cells, smooth muscle cells myofibroblasts,telocytes, or dermal epithelial cells (e.g., squamous epithelial cells)or keratinocytes, and fibroblasts. If desired, cells can be seeded priorto implanting. In some embodiments, a urinary conduit contains no stent,and in others, a stent is preferred. Urinary conduits are typicallybetween 6 and 25 mm, often greater than 10 mm in diameter.

Some of the data described below with respect to both trachea andesophagus employed tissues produced from canine (dog) cells. Hence, thematrix shown in some data is canine matrix. In addition, engineeredtrachea, esophagus, or urinary conduit may be made by culturing humancells on a substrate, that optionally is also encasing a stent. In thesecases, the final conduit contains human extracellular matrix. However,engineered airway, urinary conduit, and esophagus can be made using anymammalian or primate vascular smooth muscle cells, including humanvascular smooth muscle cells. Such primates or mammals include, withoutlimitation, pig, horse, donkey, cat, mouse, rat, cow, sheep, baboon,gibbon, and goat. Additionally, recipients of the prostheses can bewithout limitation mammals including, human, dog, pig, horse, donkey,cat, mouse, rat, cow, sheep, baboon, gibbon, and goat.

The above disclosure generally describes the present invention. Allreferences disclosed herein are expressly incorporated by reference. Amore complete understanding can be obtained by reference to thefollowing specific examples which are provided herein for purposes ofillustration only, and are not intended to limit the scope of theinvention.

Example, 1 Tracheal PGA Scaffold Preparation

A poly(glycolic) acid (PGA) sheet is cut into 5.4 cm×8.5 cm and 5.2cm×8.5 cm pieces. 5.2 cm×8.5 cm PGA mesh is rolled into a tube andinserted inside a bare-metal stent (1.7×8.5 cm). 5.4 cm×8.5 cm PGA meshis then sewn around the bare-metal stent using the absorbable PGAsuture, sandwiching the stent between the two layers of PGA mesh. Acrochet needle is applied carefully throughout the PGA/stent constructto interlace the two PGA layers together. A non-absorbable suture issewn through both ends of PGA/stent construct to suspend the constructinside a specially designed bioreactor for trachea reconstruction. Theconstruct is then dipped into 1M NaOH solution for 2 minutes to treatthe surface of the PGA mesh followed by three rinsing in distilledwater. The PGA/stent construct is then assembled in the bioreactor asshown in

Example 2 Smooth Muscle Cells (SMC) Seeding and Tracheal CultureMaintenance

Primary SMCs were isolated from dog aortas and expanded in T-75s in 20%fetal bovine serum (FBS) low glucose Dulbecco's Modified Eagle's Medium.120 million SMCs of P2 and P3 were re-suspended in 7 ml of medium andseeded onto the PGA/Stent construct inside the bioreactor, as shown inFIG. 2. The construct was cultured inside the bioreactor statically for12 weeks in 1.3 L of low glucose Dulbecco's Modified Eagle's Medium with20% FBS, basic fibroblast growth factor (10 ng/ml), platelet derivedgrowth factor (10 ng ml), L-ascorbic acid, copper sulfate, HEPES,L-proline, L-alanine, L-glycine, and Penicillin G (FIG. 2), Medium waschanged 1.5 times per week and ascorbic acid was supplemented threetimes per week.

Example 3 Decellularization of Engineered Trachea

Engineered trachea (6 cm in length) was first incubated in 250 mL CHAPSbuffer (8 mM CHAPS, 1M NaCl, and 25 mM EDTA in PBS) for 45 minutes at 37C.° under high-speed agitation, followed by thorough sterile PBSrinsing. The engineered trachea was further treated with 250 mL sodiumdodecyl sulfate (SDS) buffer (1.8 mM SDS, 1M NaCl, and 25 mM EDTA inPBS) for 45 minutes at 37 C.° with high-speed agitation. The engineeredtrachea then underwent 2 days of washing in PBS to completely remove theresidual detergent. All decellularization steps were performed understerile conditions. The decellularized engineered trachea was stored insterile PBS containing penicillin 100 U/mL and streptomycin 100 mg/mL at4 C.°.

Example 4 Esophagus PGA Scaffold Preparation

6.5 cm×10 cm PGA sheet is sewn into a cylindrical construct withabsorbable PGA suture around a compliant silicone tubing (innerdiameter=2 cm) with a suture line that is axially aligned to the PGAcylindrical scaffold. Dacron cuffs are then sewn onto the ends of thePGA tubular construct, one on each end. The construct is dipped into 1MNaOH solution for 2 minutes to treat the surface of the PGA meshfollowed by three subsequent wash in distilled water. The PGA scaffoldand silicone tubing are assembled inside a bioreactor as shown in FIG.6.

Example 5 Smooth Muscle Cells (SMC) Seeding and Pulsatile Flow Systemfor Engineered Esophagus

million dog SMCs of P2 and P3 were re-suspended in 5 ml of medium andseeded onto the PGA construct inside the bioreactor. The seededconstruct was cultured inside the bioreactor connecting to a peristalticpump, which creates cyclic radial strain of 3.0% at 1.5 Hz. Theengineered esophagus was cultured in the pulsatile culture for 10 weeksand maintained with 1.31, of low glucose Dulbecco's Modified Eagle'sMedium with 20% FBS, basic fibroblast growth factor (10 ng/ml), plateletderived growth factor (10 ng L-ascorbic acid, copper sulfate, HERPES,L-proline, L-alanine, L-glycine, and Penicillin G (FIG. 4). Half ofmedium volume was changed 1.5 times per week and ascorbic acid wassupplemented three times per week.

Example 6 Decellularization of Engineered Esophagus

The engineered esophagus was cut into two 3 cm-length pieces and werefirst incubated in 250 mL CHAPS buffer (8 mM CHAPS, 1M NaCl, and 25 mMEDTA in PBS) for either 45 or 80 minutes at 37 C.° under high-speedagitation, followed by thorough sterile PBS rinsing. The engineeredesophagus pieces were further treated with 250 mL, sodium dodecylsulfate (SDS) buffer (1.8 mM SDS, 1M NaCl, and 25 mM EDTA in PBS) for 45or 80 minutes at 37 C.° with high-speed agitation. The engineeredesophagus pieces were then washed with PBS for two days to completelyremove the residual detergent. All decellularization steps wereconducted under sterile conditions. The decellularized engineeredesophagus pieces were stored in sterile PBS containing penicillin 100U/mL, and streptomycin 100 mg/mL at 4 C.°.

Example 7 Suture Retention of Engineered Esophagus

Weights are hanged from a suture line threaded onto one side ofengineered esophagus, 2.5 to 3 mm away from the edge. Weights areincrementally added to the suture until the suture is torn from thetissue. The total weight at which the tissue is torn is recorded inunits of gram.

Example 8 Engineered Trachea for Implantation into Rat Recipient andResults of Implantations

A 4-mm diameter metal stent is encased with PGA scaffolding, sterilized,and seeded with human vascular smooth muscle cells. Thestent-scaffold-cell structure is cultured within a bioreactor for aperiod of 6-10 weeks in the presence of a nutrient culture medium. 2×10⁶P2 human smooth muscle cells (SMCs) are seeded onto the scaffoldconstructs (polygycolic acid mesh wrapped around a 4-mm diameter nitinolstent) with 4 mm diameter and 8 mm length. The tracheas were staticallysuspended on silicone tubing and cultured inside the bioreactor for 10weeks. The bioreactor medium was composed of DMEM (high glucose), bFGF(5 ng/ml), EGF (0.5 ng/ml), lactic acid (0.5 g/L), insulin (0.13 U/ml),Pen G 100 U/ml, Proline/Glycine/Alanine solution, CuSO₄ (3 ng/ml), andvitamin C (50 ng/ml). TE tracheae were cultured in 400 ml of medium atall times and only half of the medium was replaced during every mediumchange. The bioreactor medium was changed 1.5-2 times per week andvitamin C was supplemented to the culture 3 times per week. Lactic acidwas freshly added to the medium once a week. Tracheas were cultured in20% human serum for the first 4 weeks. From the 5^(th) week on, tracheaswere grown with 10% human serum.

After culture, the conduit is decellularized and stored under sterileconditions in phosphate buffered saline at 4 deg C. After several weeksof storage, the conduit is implanted into a nude rat recipient. Thechest of a 205 g nude rat was trimmed with a shaver. A 2.5 cm incisionwas made from the neck region with a pair of surgical scissor. Muscleand surrounding tissues were separated layer by layer until the tracheawas exposed. A full circumferential segment of the trachea thatconstitutes two cartilaginous rings was removed. Due to release fromtension, the gap expanded to approximately 1 cm (depending on theindividual animals). 8 mm trachea was placed in between the gap and wasanastomosed end-to-end to native trachea with at least 4 interrupted 6-0Prolene sutures for each end. Finally, the muscle and surroundingtissues were sewn together with sutures layer by layer.

At explanation after either 2 or 6 weeks of implant, the engineeredtrachea becomes invested with host epithelial cells in the lumen of theairway. During the implant time, no animals were treated withantibiotics. The implanted trachea also becomes invested with other hostcells including fibroblasts, and also becomes invested with hostmicro-vasculature both in the wall of the engineered trachea and in thelumen of the engineered trachea. The dense and rapid influx ofmicrovasculature (seen as early as 2 weeks after implantation, byhistological evaluation) aids in resistance to infection, since hostleukocytes can easily gain access to the implanted tissue to fight anyinfecting organisms. Over the longer term, the engineered trachea mayalso become invested with cartilaginous cells of the native trachea, aswell as smooth muscle cells that occupy the native tracheal wall andother airways. The implanted tracheas all resisted dilatation, rupture,and perforation, which could lead to device failure and to infection inthe animal. In addition, the engineered implanted tracheas did not showany evidence of either immune rejection, or of bacterial or fungalinfection, during the entirely of the implantation period. The implantedtrachea may resist stenosis or scarring which limits air flow to thelungs.

Engineered, decellularized human tracheas were explanted from ratrecipients at two week and six weeks. After two weeks of implantation,robust tissue formation was observed in the lumen of the engineeredtracheas, with evidence of extensive microvascularization. Also aftertwo weeks, luminal tissue was immunostained and was strongly positivefor cytokeratin-14, an epithelial marker. See FIGS. 17 and 18.

After 6 weeks of implantation, engineered tracheas displayed goodincorporation into host tissues, with formation of fibrous tissuesurrounding the implanted engineered trachea, some evidence of residualdecellularized human matrix, as well as ingrowth of luminal tissue.There was also some evidence of host cell infiltration into thepreviously acellular matrix of the tracheal implant. The implanted,engineered trachea was physically intact, without evidence ofdistension, perforation, or anastomotic breakdown. No evidence ofexcessive leukocyte infiltration or infection was observed in explantedspecimens. See FIG. 19.

These results overall show that producing engineered, decellularizedtracheas is feasible. Engineered tracheas can be sutured into recipientairways and can conduct air and allow the recipient to survive for longtime periods. Engineered tracheas do not exhibit evidence of infectionafter implantation, and rapidly become invested with host cells andtissues and microvasculature after only a few weeks. Cells and tissuethat infiltrates the engineered tracheas is highly vascular, and alsocontains cells that are native to the respiratory system (pulmonaryepithelium). Engineered tracheas remain mechanically robust and do notsuffer from mechanical failures such as perforation, dilatation,rupture, or anastomotic breakdown.

Example 9 Urinary Conduits

Based on methods pioneered by Dahl, Niklason, and colleagues [6-10], wehave developed methods to grow tubular engineered tissues from bankedhuman smooth muscle cells (SMC) that are seeded onto a biodegradablescaffold and cultured in bioreactors. No cells are harvested from therecipient for this process. After 10 weeks of culture, the engineeredtissues are comprised of SMC and the extracellular matrix they haveproduced, which is primarily type I collagen. These tissues are thendecellularized, creating an acellular tubular tissue that has excellentmechanical characteristics (rupture strengths >2,000 mm Hg) [10]. Wehave tested these tubular engineered tissues as arteriovenous grafts ina baboon model, and they have shown excellent function,biocompatibility, zero mechanical failures, and zero infection.

TABLE 1 Suture Burst Press, Strength, g mmHg 6-mm diameter conduit 178 ±11 3337 ± 343 (n = 37) (n = 10) 6-mm conduit, stored 12 170 ± 22 2651 ±329 months in PBS buffer (n = 9) (n = 5)

We believe that the acellular engineered tissues will mitigate many ofthe complications that are associated with ileal conduits. Because ourtissues are non-living and repopulate gradually with host cells, conduitischemia and the associated mechanical failures will be extremelyunlikely. Because our tissues do not actively absorb electrolytes, theyshould not cause a metabolic acidosis. Because our conduits do notfoster the growth of commensal bacteria, they should not triggerrecurrent urinary tract infections, And because they are availableoff-the-shelf, complications due to bowel resection will be avoided. Ouracellular tubular engineered tissues have many favorable properties thatmay make them superior to segments of small intestine for urinarydiversion. Since our urinal); conduit is pre-manufactured using bankedcells and can be stored on the shelf, there is no need to resect asegment of intestine from the patient—surgery on the bowel is completelyavoided. Since our conduit is non-living, there is essentially no riskof tissue ischemia after implantation. Rather, host cells graduallymigrate into the acellular matrix, with formation of commensuratemicrovasculature. Since our conduit does not actively absorb its luminalcontents, the risk of hyperchloremic metabolic acidosis is substantiallyreduced. And, since our conduit does not harbor intestinal flora, therisks of recurrent urinary tract infections should be markedly reduced,Hence, essentially all of the common complications associated with useof an ileal conduit could be reduced or Obviated by our acellularengineered tissues [11].

Approximately 10,000 cystectomies are performed annually in the US, withbladder cancer being the leading indication. In patients with T1 diseaserefractory to conservative measures and in patients with T2 tumors,surgical removal of the bladder with possible resection of associatedpelvic organs remains the contemporary standard of care [12]. Other,less common reasons for cystectomy include neurogenic bladder (when itthreatens renal function), severe radiation injury to the bladder, andintractable incontinence as well as chronic pelvic pain syndromes infemales. All currently available surgical options for construction ofurinary diversions involve the use of a segment of small or largeintestine (FIG. 11). Though it is possible to build more complex,continent reservoirs, the majority of patients in North Americaundergoing cystectomy are reconstructed using the ilea conduit technique[13].

Shabsigh [14] reported that within 90 days of surgery, gastrointestinalcomplications occurred most commonly (29%), followed by infections(25%), wound related complications (15%), cardiac (11%), andgenitourinary complications (11%). Electrolyte abnormalities,particularly metabolic acidosis, occur in 70% of patients, though oftenof unknown clinical significance. Severe electrolyte disturbances occurin 10% of patients with an ileal conduit [3, 5]. Osteomalacia can resultfrom chronic acidosis with consequent release of calcium from bones.Acute pyelonephritis occurs in 10-17% of patients with colon and ilealconduits, and 4% of patients with ileal conduits die of sepsis [15],Cancer occurs in ileal conduits—anaplastic carcinoma and adenomatouspolyps have been described. The reported rate for cancer in ilealconduits varies from 6-29% of all patients, though cancers can takedecades to develop [5]. Early bowel complications typically consist ofanastomotic leaks, enteric fistulas, bowel Obstruction, and prolongedileus [11]. Bowel obstruction has been reported in as many as 5-10% ofpatients, with the majority responding well to conservative treatmentwhile approximately 3% require surgery. Bowel anastomotic leak is apotentially devastating complication reported in 1-5% of patients, whichcan lead to abscess formation, peritonitis, and sepsis [5],

Example 10 Urinary Conduit

A urinary conduit is cultured using human smooth muscle cells that arecultured on a tube of PGA mesh scaffold in a bioreactor as described.After a culture period of 6-10 weeks, the resulting tubular tissue isdecellularized, and then stored in phosphate buffered saline at 4 deg C.for a period of several months. Thereafter, a cynomolgus monkey (whichis an old-world primate that is phylogenetically close to humans and istherefore unlikely to reject the human engineered tissue) is preparedfor implantation of the urinary conduit. After induction of anesthesia,a laparotomy is performed and the ureters of both kidneys are isolatedand excised from the bladder wall, which is oversewn. The ureters areanastomosed to the urinary conduit, the other end of which isanastomosed to the abdominal wall to allow urine to flow from theureters, through the conduit, and outside the animal's body, Aftercompletion of the implantation, the abdomen is closed and the animal isrecovered from anesthesia. Thereafter, the urinary conduit is seen toconduct urine to the outside of the body to a collecting bag. There isno evidence of leakage of urine into the abdomen or from the anastomoseswith the abdominal wall or the ureters.

An implanted, engineered urinary conduit may become invested on theluminal surface with urinary epithelium. The implanted urinary conduitmay become invested with fibroblasts in the wall of the conduit, maybecome invested with micro-vasculature which contributes to resistanceto infection, and may become invested with smooth muscle cells similarto urinary bladder. The implanted, engineered urinary conduit may resistinfection with skin flora and with organisms from the urinary tract. Theimplanted, engineered urinary conduit may resist scarring andconstriction which would impede urine flow, it may resist dilatationwhich would cause pooling of urine in the conduit, it may resist kinkingand obstruction which would impede urine flow, it may resist formationof intra-abdominal adhesions which can obstruct the conduit orintestinal tissues, and it may resist the creation of hyperchloremicmetabolic acidosis in the host animal.

REFERENCES

The disclosure of each reference cited is expressly incorporated herein.

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1. An artificial airway for replacement of damaged tissue byimplantation into a respiratory tract of a recipient, said artificialairway comprising: a tubular stent, and substantially acellular,non-layered, contiguous, extracellular matrix surrounding the stent onits inner and outer surfaces.
 2. The artificial airway of claim 1wherein the extracellular matrix is rotationally fixed with respect tothe stent.
 3. The artificial airway of claim 1 wherein the extracellularmatrix on the outer surface is rotationally fixed with respect to thestent and with respect to the extracellular matrix on the inner surface.4. The artificial airway of claim 1 further comprising acellularextracellular matrix linking the acellular extracellular matrix on theinner and outer surfaces.
 5. The artificial airway of claim 1 whereinthe extracellular matrix is populated with tracheal cells selected fromthe group consisting of epithelial cells, cartilage cells, endothelialcells, smooth muscle cells, and fibroblasts.
 6. The artificial airway ofclaim 5 wherein the tracheal epithelial cells are autologous to therecipient.
 7. The artificial airway of claim 1 wherein the stent ismetal.
 8. The artificial airway of claim 1 wherein the stent ispolymeric.
 9. The artificial airway of claim 1 wherein the extracellularmatrix was produced and secreted by vascular smooth muscle cells. 10.The artificial airway of claim 9 which comprises fragments of apolyglycolic acid mesh on which the vascular smooth muscle cells werecultured.
 11. The artificial airway of claim 1 wherein greater than 20%of dry tissue weight of the artificial airway is collagen.
 12. Theartificial airway of claim 1 wherein the extracellular matrix is between200 and 700 microns in thickness.
 13. The artificial airway of claim 1which is implanted in the respiratory tract of the recipient, whereinthe recipient is a mammal.
 14. The artificial airway of claim 13 whichis impermeable to liquid and gas.
 15. A method of making an artificialairway, comprising the steps of: encasing a tubular stent having aninner and outer surface in at least two layers of a mesh scaffold, afirst of the at least two layers on the interior surface and a second ofthe at least two layers on the exterior surface; seeding the meshscaffold with vascular smooth muscle cells and culturing the vascularsmooth cells on the mesh scaffold in a bioreactor for 6-10 weeks,whereby the smooth muscle cells proliferate and secrete extracellularmatrix on the mesh scaffold; decellularizing the tubular stent to form asubstantially acellular tubular airway stent encased in extracellularmatrix on the inner and outer surfaces.
 16. The method of claim 15wherein the mesh scaffold is biodegradable.
 17. The method of claim 15wherein the mesh scaffold degrades during the steps of culturing anddecellularizing.
 18. The method of claim 15 wherein the mesh scaffoldcomprises polyglycolic acid.
 19. The method of claim 15 wherein thesmooth muscle cells are isolated from an aorta.
 20. The method of claim15 wherein the decellularization step employs agitation.
 21. The methodof claim 15 wherein the decellularization step employs detergenttreatment.
 22. The method of claim 15 wherein the artificial airway isstored at about 4 deg C. prior to implantation in a recipient.
 23. Themethod of claim 15 further comprising the step of interlacing the innerand outer layers of mesh scaffold at a plurality of locations along thelength and/or circumference of the stent.
 24. An artificial esophagusfor replacement of damaged tissue by implantation, said esophaguscomprising: substantially acellular extracellular matrix formed as atube of greater than 10 mm diameter, wherein the artificial esophagushas a suture retention of greater than 150 grams.
 25. The artificialesophagus of claim 24 which is connected to a digestive tract and isimpermeable to food, liquid, and gas.
 26. The artificial esophagus ofclaim 25 which is populated with esophageal cells selected from thegroup consisting of epithelial cells, endothelial cells, smooth musclecells, and fibroblasts.
 27. The artificial esophagus of claim 24 whereinthe extracellular matrix was produced and secreted by vascular smoothmuscle cells.
 28. The artificial esophagus of claim 24 wherein greaterthan 30% of dry tissue weight of the artificial esophagus is collagen.29. The artificial esophagus of claim 24 whish has a rupture strength ofgreater than 1000 mm Hg.
 30. The artificial esophagus of claim 24 whichcomprises a tubular stent.
 31. An artificial urinary conduit forimplantation in a patient in need of urinary diversion and drainage,comprising: a tubular, substantially acellular, extracellular matrixformed as a tube of greater than 10 min diameter, wherein theextracellular matrix was produced and secreted by non-autologous smoothmuscle cells, wherein the artificial urinary conduit has a rupturestrength of greater than 1000 mm Hg.
 32. The artificial urinary conduitof claim 31 which is connected to a ureter of a patient and drainsthrough a stoma in skin of the patient.
 33. The artificial urinaryconduit of claim 31 which has a suture retention strength of greaterthan 150 grams.
 34. The artificial urinary conduit of claim 31 which ispopulated with cells selected from the group consisting of urinaryepithelial cells, endothelial cells, smooth muscle cells, andfibroblasts.
 35. An artificial stented urinary conduit for implantationin a patient in need of urinary diversion and drainage, comprising: atubular, substantially acellular, extracellular matrix formed as a tube,wherein the extracellular matrix was produced and secreted bynon-autologous smooth muscle cells, wherein the artificial urinaryconduit comprises a tubular stent and has a rupture strength of greaterthan 1000 mm Hg.
 36. The artificial urinary conduit of claim 35 whichhas a suture retention strength of greater than 150 grams.
 37. Theartificial urinary conduit of claim 35 which is populated with cellsselected from the group consisting of urinary epithelial cells,endothelial cells, smooth muscle cells, and fibroblasts.